1. Field of the Invention
This invention relates to novel CLAVATA3/ESR (CLE) genes, GrCLEs, cloned from the potato cyst nematode Globodera rostochiensis, specifically expressed in the dorsal esophageal gland cell of nematode parasitic stages, Gr-CLE proteins with multiple CLE motifs, and a method of using host-derived RNA interference (RNAi) of Gr-CLE genes to generate novel resistance against G. rostochiensis in transgenic potatoes.
2. Description of the Relevant Art
Potato cyst nematodes (G. rostochiensis and G. paffida) are obligate root parasites that have evolved highly sophisticated parasitic relationships with specific host plants (Davis et. al. 2004. Trends Parasitol. 20:134-141; Hussey and Grundler. 1998. In: Physiology and Biochemistry of Free-Living and Plant-Parasitic Nematodes. R. N. Perry and D. J. Wright, eds. CAB International Press, Oxford, Pages 213-243). Infective second-stage juveniles (J2) hatch from eggs within the cyst in the soil and infect host roots. The J2 uses its stylet (hollow mouth spear) to aid its intracellular root migration and selects an initial cell within root tissues for the development of complex feeding structure called a syncytium (Dropkin, V. H. 1969. Annu. Rev. Phytopathol. 7:101-122.; Jones, M. G. K. 1981. Ann. Appl. Biol. 97:353-372). Juveniles of G. rostochiensis preferentially select a fully differentiated cortical cell in potato roots to initiate a syncytium (Jones and Northcote. 1972. J. Cell Sci. 10:789-809). At the onset of feeding, the nematode becomes sedentary and relies on the syncytium for nourishment for the remainder of its life cycle.
The formation of the syncytium represents one of the most complicated plant responses triggered by plant pathogens. The syncytium is a highly metabolically active multinucleate structure formed by extensive cell wall dissolution of neighboring cells around the initial syncytial cell. Its characteristics include enlarged nuclei and nucleoli, dense cytoplasm, increased numbers of subcellular organelles, small vacuoles, and thickened cell walls with elaborate wall ingrowths formed adjacent to the vascular tissues (Jones 1981, Ann. Appl. Biol. 97:353-372supra). The formation of wall ingrowths facilitates nutrient uptake from the xylem into the syncytium to meet the demands of the feeding nematode. Some of these developmental characteristics are features of various types of plant cells, inducing meristematic cells and transfer cells, suggesting that cyst nematodes have likely evolved mechanisms to manipulate plant developmental pathways to form a novel cell type (Mitchum et al. 2008. Curr. Opin. Plant Biol. 11:75-81).
Secreted proteins encoded by parasitism genes expressed within the single dorsal and two subventral esophageal gland cells of cyst nematodes represent the major functional molecules that directly or indirectly modulate plant cellular processes involved in the induction, formation, and maintenance of the syncytium (Davis et al. 2004, supra; Davis et al. 2008. Curr. Opin. Plant Biol. 11:360-366; Hussey, R. S. 1989. Annu. Rev. Phytopathol. 27:123-141.). Parasitism genes from the soybean cyst nematode (Heterodera glycines) encoding small secreted proteins with similarity to CLAVATA3/ESR-related (CLE) signaling peptides were identified previously (Gao et al. 2003. Mol. Plant-Microbe Interact. 16:720-726.; Olsen and Skriver. 2003. Trends Plant Sci. 8:55-57; Wang et al. 2001. Mol. Plant Microbe Interact. 14:536-544). This is the only known report of CLE genes outside the plant kingdom. Plant CLEs encode small proteins with an N-terminal signal peptide, a variable domain, and a conserved 14-amino acid (aa) domain called the CLE motif located at or near their C-termini (Cock and McCormick. 2001. Plant Physiol. 126:939-942). Plant CLEs have been suggested to have roles in shoot, floral, and root meristem maintenance, organ size regulation, apical dominance, and vascular development (Casamitjana-Martinez et al. 2003. Curr. Biol. 13:1435-1441; Fiers et al. 2004. Gene 327:37-49; Fiers et al. 2005. Plant Cell 17:2542-2553; Hirakawa et al. 2008. Proc. Natl. Acad. Sci. USA 105:15208-15213; Hobe et al. 2003. Dev. Genes Evol. 213:371-381; Ito et al. 2006. Science 313:842-845; Strabala et al. 2006. Cell 100:635-644). To date, more than one hundred putative CLE genes have been identified from diverse plant species (Oelkers et al. 2008. BMC Plant Biol. 8:1.). Sequence alignments among plant CLE proteins reveal little sequence similarity outside their conserved CLE domains and mounting evidence implicates the conserved CLE domain as the major functional domain of CLE proteins (Fiers et al. 2005, 2006., supra; Hirakawa et al., supra; Ito et al., supra; Kondo et al. 2006. Science 313:845-848; Ni and Clark. 2006. Plant Physiol. 140:726-733). More recently, plant CLEs that encode multiple tandem C-terminal CLE motifs (Kinoshita et al. 2007. Plant Cell Physiol. 48: 1821-1825; Oelkers et al., supra) have been identified, although the biological significance of the repeated CLE motifs and the function of this novel class of plant CLE genes are yet to be discovered.
The Arabidopsis genome contains 32 CLE genes that encode CLE proteins with a single CLE motif located at or near their C-termini (Cock and McCormick, supra). The founding member, CLAVATA3 (CLV3), functions as a peptide ligand that interacts with the CLV1/CLV2 receptor complex to signal a stem cell restricting pathway in shoot and floral meristems (Fletcher et al. 1999. Science 283:1911-19). CLV1, expressed in the central zone of the shoot apical meristem (SAM), encodes a membrane-bound leucine-rich repeat receptor-like kinase (LRR-RLK; Clark et al. 1997. Cell 89:575-585). CLV2, having a much broader expression pattern than that of CLV1, encodes a LRR receptor-like protein lacking a kinase domain (Jeong et al. 1999. Plant Cell 11:1925-1933). The functional form of CLV3 is a 12-aa peptide derived from its CLE domain (Kondo et al., supra). The physical interaction between the dodeca-CLV3 peptide and the LRR domain of CLV1 was also demonstrated (Ogawa et al. 2008. Science 319:294). More recently, it was revealed that CORYNE (CRN), a novel receptor kinase, is a new component of the CLV3 signaling pathway in Arabidopsis (Muller et al. 2008. Plant Cell 20:934-946). The Arabidopsis WUSCHEL (WUS) gene, which encodes a homeodomain transcription factor, is also an important regulator that promotes stem cell identity in the SAM (Laux et al. 1996. Development 122:87-96; Mayer et al. 1998. Cell 95:805-815). WUS is a key target of the CLV signaling pathway and a negative regulatory feedback loop between WUS and CLV genes controls the size of the stem cell population in the SAM (Brand et al. 2000. Science 289:617-619; Schoof et al. 2000. Cell 100:635-644). Mutations in any of the CLV genes result in enlarged shoot and floral meristems due to the uncontrolled proliferation of stem cells (Clark et al. 1993. Development 119:397-418, Clark et al. 1995. Development 121:2057-2067; Fletcher et al, supra; Kayes and Clark. 1998. Development 125:3843-3851), whereas CLV3 overexpression or mutation of the WUS gene terminates SAM development (Brand et al. 2000, supra; Laux et al., supra).
Surprisingly, overexpression of a H. glycines CLE gene (Hg-4G12) in Arabidopsis caused premature termination of the SAM similar to CLV3 (Davis, in press; Wang et al. 2005. Mol. Plant Pathol. 6:187-191). In addition, the nematode CLE was able to rescue the Arabidopsis clv3-1 mutant phenotype when expressed under the control of the CaMV 35S promoter (Wang et al. 2005, supra). These results suggested that nematode and plant CLES share functional similarity and led to the hypothesis that ligand mimicry of plant CLE signaling peptides may be an important mechanism in cyst nematode parasitism of host plants (Mitchum et al., supra; Wang et al. 2005, supra).
Plant-mediated RNA interference (RNAi) has been used to target nematode parasitism genes and helped attain broad resistance against four root-knot nematode species in the model plant Arabidopsis (Huang et al. 2006. Proc. Natl. Acad. Sci. USA 103: 14302-14306). The double-stranded (dsRNA) or small interfering (siRNA) molecules were taken up by the nematode from soaking solution (in vitro) or from plant tissue (in planta). RNAi has been observed to function in both cyst and root-knot nematode species (Lilley et al. 2007. Molecular Plant Path. 8: 701-711). Production of parasite-specific dsRNA in plant cells has been suggested as a novel and durable strategy for control of plant parasitic nematodes including cyst nematodes (e.g. Gheysen and Vanholme. 2007. Trends in Biotech. 25: 89-92; Steeves et al. 2006. Func. Plant Biol. 33: 991-999; Sindhu et al. 2008. Journal of Experimental Botany 60:315-324; Patel et al. 2008. Journal of Nematology 40:299-310).
The use of nematode resistant cultivars is the most economical and environmentally-safe means of nematode control; therefore, there is a need for G. rostochiensis- and G. pallida-resistant cultivars.